EP1307292B1

EP1307292B1 - Highly parallel fabrication of microarrays by ink jet printheads

Info

Publication number: EP1307292B1
Application number: EP01954138A
Authority: EP
Inventors: Howard John Manning
Original assignee: Arrayjet Ltd
Current assignee: Arrayjet Ltd
Priority date: 2000-08-03
Filing date: 2001-08-02
Publication date: 2005-12-28
Anticipated expiration: 2021-08-02
Also published as: DE60116326T2; WO2002011889A1; ATE314148T1; US20040145631A1; AU2001276486B2; EP1307292A1; ES2256275T3; DE60116326D1; GB0018963D0; US7128393B2; AU7648601A; CA2414457A1

Abstract

A method is described of operating an ink jet printhead having one or more manifolds each connected to more than one chamber, each chamber being associated with a nozzle and capable of ejecting drops therefrom: a number of different liquids larger than the number of manifolds is introduced into the printhead via the nozzles; and the volume of the liquid subsequently printed from each nozzle is less than the volume of the chamber associated with that nozzle, and also less than the volume of liquid introduced into that chamber. By this means the printhead prints more different liquids than is conventionally possible without mixing them.

Description

This invention relates to the use of ink jet printers to make biological microarrays.

Advances in biological and chemical science are demanding the testing of large numbers of samples in parallel. For example, the sequencing of human and animal genomes has created a need to determine the function of genes through expression studies. In this field, in pharmacogenomics and toxicology screening, and in many other applications, there is a need to test a large number of interactions between probe and target.

A technology which has emerged to address this need is the microarray, also known as the DNA microarray or biochip, and by other terminology. This consists of a substrate on which a compact array of biological or chemical samples, known as probes, is immobilised. The microarray is exposed to a sample, known as the target, which is to be tested against the probes. The interactions are recorded by suitable instrumentation and the data is manipulated.

Microarrays are made at present by two methods: the probes can be synthesised on the array, by applying constituents of the probes to build them up in situ; or pre-synthesised probes can be spotted onto the array. This invention relates to the latter method.

The task of spotting a microarray consists of transferring extremely small amounts of many different liquids from separate reservoirs to closely spaced positions on a number of microarrays. There may be anything from tens of different liquids to hundreds of thousands of them, supplied typically in multiple 96, 384 or 1536-well microtitre plates. Some tens or hundreds of substrates need to be spotted with each of the liquids; typical spot volumes are of the order of a nanlolitre, and spots may be separated by a few hundred microns.

Spotting is achieved at present in two main ways: in the first method, pins are dipped into the wells to pick up samples of the liquids, and then moved on a three-axis transport to touch the substrates and deposit drops. Several pins may be used in parallel to speed up the spotting.

There are disadvantages to this technology: the pins have to be washed and dried before picking up samples of another set of liquids. The pins have to touch the substrate, which requires high precision, carries a risk of damage, and is slow. The volume of liquid spotted is rather large, is not well controlled and cannot be varied easily. The configuration of spots on the microarray corresponds to the arrangement of liquids in the wells, as the pins are all brought into contact with the substrate simultaneously. A considerable proportion of each liquid is wasted.

The second method of spotting is to project the liquid through the air onto the substrate, without contact. In principle, ink jet printing technology is eminently suitable: it produces small droplets, very reproducibly, and positions them accurately on the substrate. In some cases, the droplets are sufficiently small that multiple droplets can be applied to a given spot to vary its volume. Ink jet printing is very rapid, and is entirely flexible as to what liquid is deposited where on the substrate.

The main difficulty with ink jet technology is that, although some printheads have large numbers of nozzles, they are designed to print typically one or four colours of ink. Their inlets lead to manifolds which connect many chambers, each associated with a nozzle. If such a printhead is applied conventionally to the manufacture of microarrays, the speed of the process is limited by the fact that only one or four liquids is handled at a time, and the fact that there are many nozzles is of little help. The printing itself is very quick, and it is the process of emptying and refilling the printhead which determines the overall manufacturing time.

Other difficulties with ink jet printheads are: some use local boiling of the liquid to eject drops, which could damage some biological samples; others are constructed from materials incompatible with the chemicals to be printed onto microarrays; some are designed for office printers, and are unsuitable for third party integration into industrial systems; and others are designed for industrial use, but require large volumes of liquid to operate.

For the reason given above, standard ink jet printheads are not used in the manufacture of microarrays; rather adapted printheads or devices akin to printheads, are used instead. These do not take advantage of the manufacturing capabilities of ink jet companies, and do not handle large numbers of liquids.

Examples of such devices are disclosed in WO 98/45205A and US 6 083 763 A.

The present invention relates to a way of using standard ink jet printheads to handle a number of different liquids larger than the number of colours it is designed to print, without a mixture of the liquids being printed.

In accordance with a first aspect of the present invention there is provided printing apparatus capable of printing a number of different samples without the samples being mixed, as defined in claim 1.

According to a second aspect of the present invention there is provided a method of printing a number of different samples using the apparatus of the first aspect, as defined in claim 12. Further embodiments are defined in the dependent claims.

A commercially promising application of the invention is to print the liquids as spots of picolitre to nanolitre volume onto a substrate for the production of biological or chemical microarrays. An advantage of ink jet printheads is that they can print while there is relative motion between the printhead and the substrate, increasing speed of production.

A specific embodiment of the invention will now be described, purely by way of example, with reference to the accompanying drawings, in which:

Figure 1 shows a side cross-sectional view of a suitable ink jet printhead;

Figure 2 shows a front cross-sectional view of a suitable ink jet printhead; and

Figure 3 shows the liquids being introduced into the printhead from a microtitre plate by means of a connection block.

Referring to Figures 1 and 2, each nozzle 1 is associated with a long narrow chamber 4 excavated within lower component 3. The chambers open out at the rear into manifolds 5 each serving multiple chambers. There are typically one or four manifolds 5, each fed by a supply via a filter 6.

Drops are ejected when the walls between the chambers 4 are deflected, causing pressure waves within the chambers 4. The length of the chambers 4, defined by cut-outs in the upper component 2, determines the drop size ejected. If the chambers 4 are long (6mm in a particular printhead), relatively large drops (typically 50 picolitres) are ejected, which are suitable for binary printing and also for the production of microarrays. If the chambers 4 are short (1 mm in another printhead), small drops (typically 7 picolitres) are produced; then multiple drops can be used for greyscale printing, or for producing microarray spots of controllable size.

In the case of the binary printhead, the chambers 4 are long compared with their lateral dimensions (typically 75 microns by 390 microns), so the liquid tends to advance along the chambers 4 towards the nozzles 1 as printing proceeds; there is little tendency for liquid at the rear of a chamber 4, or that entering the chamber 4 from the manifold 5, to mix with liquid near the nozzle 1. The pathway for diffusion to introduce into a chamber 4 liquid from another chamber 4, via the connecting manifold 5, is long and unfavourable for mixing. Therefore nearly the entire contents of a chamber 4 can be printed without contamination by liquid from elsewhere.

Referring to Figure 3, a possible embodiment of the invention involves a connection block 10 interposed between the printhead 9 and a microtitre plate 11 to allow multiple liquids to be introduced into the printhead via the nozzles 1. In a preferred embodiment the connection block 10 would include a filter layer.

The connection block 10 has moulded rubber seals 12 which separate multiple regions 13 (typically 48 in number) of the printhead, each containing several nozzles 1 (typically seven, with three blocked by the seal 12).

Capillaries 14 project downwards from the regions 13 into the wells 15 of a microtitre plate. The pitch of the wells 15 (typically 4.5mm for a 384-well plate) is larger than that of the regions 13 (typically ten times the pitch 141 microns of the nozzles), so there need to be multiple (typically three) rows of capillaries 14; only one row is shown in Figure 3.

The printhead 9 may initially be full of a neutral liquid. Suction is applied at point 8 until samples have been drawn into the printhead 9, slightly more than filling the corresponding chambers. The nozzles may act as restrictors to control the flowrate during filling. If the nozzles are of small diameter at their exit faces than internally, they resist ingress of any dirt particles sufficiently large to block nozzles subsequently. The connection block 10 can be equipped with a course filter to minimise the population of dirt particles entering the printhead 9.

An alternative embodiment would have the sample being forced into nozzles 1 from the wells 15. This could be done by pressurising the wells 1, which may be provided with self-sealing covers, so that the liquid is pushed out of them into the nozzles 1; alternatively seals on the lower surface of the connection block could isolate the wells. Sealing the wells 1 would have the benefit that different wells 1 could be placed under different pressures so that different amounts of sample could be pushed into the nozzle 1; the seals could also guard against dirt from the atmosphere getting into the printhead. Another way in which samples could be forced into the nozzles 1 is by placing the samples in a preloaded cartridge comprising of reservoirs equipped with pistons which push samples out of the reservoirs when required.

As soon as the liquids have been introduced into the printhead 9, it is detached from the connection block 10, wiped and moved by means of an x-y-z motion control to the microarrays to be spotted. The amount of liquid printed from each nozzle 1 is less than the volume of the corresponding chamber 4, so the mixture of liquids in the manifolds 5 is not printed. The timescale of the printing (seconds) does not allow diffusion to contaminate one chamber 4 with the liquid from another. In a preferred embodiment, the row of nozzles 1 is parallel to the direction of relative motion during printing, as this would allow multiple drops to be placed at one point, increasing the amount of liquid at that point.

If the filling and printing are well controlled, the fraction of the liquid drawn from the wells 15 which is wasted should be substantially less than half.

After spotting, the printhead 9 may be taken to a filling station and neutral liquid drawn in through the nozzles. Then another set of liquids can be charged into the printhead 9 and spotted. The use of neutral liquid prevents contamination of the liquid in a chamber 4 by residues of liquids previously introduced into it. Perfect displacement of the liquid in a chamber 4 by neutral liquid entering via its nozzle 1 is impossible, so the volume of neutral liquid introduced into each nozzle 1 should be several times the volume of the chamber 4 associated with that nozzle 1. When the next set of liquids is introduced, they will be diluted slightly by the neutral liquid present in each chamber 4, however, the dilution will be very small and consistent.

At no stage does the printhead 9 have to be dried out, and air never enters the printhead 9.

Only one nozzle 1 is needed to print the liquid in each region 13. The fact that several nozzles 1 are charged with each liquid means that occasional nozzle blockages or other failures do not limit the lifetime of a printhead 9 in the system. Automated testing of nozzle failures would allow the system to switch to alternative nozzles 1.

The time taken to spot a complex microarray by conventional means is dominated by the speed of the x-y-z motion and the loading of the printhead. Ink jet printers are capable of printing while the printhead is in motion, or the substrate is moving relative to the printhead; and the present invention allows the printhead to be loaded with multiple liquids without emptying and drying the printhead. These advantages lead to a substantial speed improvement relative to mechanical spotting systems.

Claims

Printing apparatus comprising a means for ejecting samples, capable of ejecting a number of different samples without the samples being mixed; characterised by the means for ejecting samples comprising at least one manifold connected to a plurality of chambers, each chamber associated with at least one nozzle, and means for introducing samples into the chambers via the nozzles, wherein the apparatus is able to eject a number of different samples larger than the number of manifolds.
Printing apparatus as in Claim 1, wherein the chamber is longer in the direction of sample motion during printing than in a perpendicular direction to the direction of sample motion during printing.
Printing apparatus as described in any of the previous Claims, wherein the means for ejecting samples is full of fluid at the outset of printing.
Printing apparatus as in Claim 3, wherein the fluid is a liquid.
Printing apparatus as in Claims 1 to 3, wherein the means for ejecting samples is full of a solid at the outset.
Printing apparatus as in Claim 5, wherein the solid is a weak solid which has deformable properties.
Printing apparatus as in any of the previous Claims, where the means for ejecting samples further comprises a connection block attached to the nozzles.
Printing apparatus as in Claim 7, wherein the connection block comprises seals which act against a nozzle plate to separate different samples.
Printing apparatus as in Claims 7 and 8, wherein the connection block comprises a filter layer.
Printing apparatus as in any of the previous Claims, wherein the means for ejecting samples is attached to a moving means which allows samples to be taken into the means for ejecting samples at one point and expelled at a second point or points.
Printing apparatus as in Claim 10, wherein nozzles on the means for ejecting samples are positioned so that they are parallel to the motion during printing.
A method of printing a number of different samples using the printing apparatus described in any of the previous Claims.
A method of printing a number of different samples, as described in Claim 12, wherein a number of different samples larger than the number of manifolds is introduced into chambers via the nozzles in the means for ejecting samples.
A method of printing a number of different samples, as claimed in Claims 12 or 13, wherein the volume of sample printed from each nozzle is less than the volume of the chamber associated with that nozzle.
A method of printing different samples, as described in Claims 12 to 14, wherein the volume of the sample printed from each nozzle is less than the volume of sample introduced into the chamber.
A method of printing different samples, as claimed in Claims 12 to 15, wherein the volume of sample introduced into each nozzle is greater than the volume of the chamber associated with that nozzle.
A method of printing different samples, as described in Claims 12 to 14, wherein printing is carried out within a time after the introduction of different samples less than the time taken for diffusion to contaminate the sample in any chamber with sample from any other chamber via the manifold connecting them.
A method of printing different samples, as described in Claims 12 to 17, wherein the samples introduced via the nozzles displace an initial fluid contained in the nozzle towards and into the manifolds.
A method of printing different samples, as described in Claims 12 to 18, wherein the samples are introduced into the nozzles via the application of suction to the manifolds.
A method of printing different samples, as described in Claims 12 to 18, wherein the samples are introduced into the nozzles by the application of pressure at the nozzles or to the connection block.
A method of printing different samples, as claimed in Claim 20, wherein there are provided sample reservoirs which are provided with a penetrable seal.
A method of printing different samples, as described in Claims 20 and 21, wherein the sample wells are each pressurised separately.
A method of printing different samples, as claimed in Claims 20 to 22, wherein the reservoirs may be pressurised using pistons.
A method of printing different samples, as claimed in Claims 12 to 18, wherein samples are introduced into the nozzles by the actuation in reverse of the means for ejecting samples.
A method of printing different samples, as described in Claims 12 to 24, wherein the volume of each sample printed is a high proportion of the volume that was introduced into that chamber.
A method of printing different samples, as described, in Claims to 12 to 25, wherein the total volume of a given sample printed can be increased above the volume introduced into the means for ejecting samples by:

(a) introducing a first set of samples and printing; and

(b) introducing and printing a second and subsequent set of samples.
A method of printing different samples, as described in Claims 12 to 26, wherein successive sets of samples may differ, so that the number of different samples that may be printed can be increased, potentially above the number of chambers in the means for ejecting samples.
A method of printing different samples, as described in Claim 27, wherein between the printing of a first set of samples and the introduction of a next set of samples, the means for ejecting samples is cleaned by the introduction of a neutral liquid into the means for ejecting samples.
A method of printing different samples, as described in Claims 12 to 28 wherein a high proportion of the sample introduced into the printing apparatus is printed.
A method of printing different samples, as described in Claims 12 to 29, wherein the samples are able to be printed while the means for ejecting samples is moving.
A method of printing different samples, as described in Claims 12 to 30 which can be used to produce microarrays.
Printing apparatus as claimed in Claims 1 to 11, which can be used to produce microarrays.